Genetic
engineering, also known as recombinant DNA technology, means altering the
genes in a living organism to produce a Genetically Modified Organism (GMO)
with a new genotype. Various kinds of genetic modification are possible:
inserting a foreign gene from one species into another, forming a transgenic
organism; altering an existing gene so that its product is changed; or
changing gene expression so that it is translated more often or not at all.

Genetic
engineering is a very young discipline, and is only possible due to the
development of techniques from the 1960s onwards. Watson and Crick have made
these techniques possible from our greater understanding of DNA and how it
functions following the discovery of its structure in 1953. Although the final
goal of genetic engineering is usually the expression of a gene in a host, in
fact most of the techniques and time in genetic engineering are spent isolating
a gene and then cloning it. This table lists the techniques that we shall look
at in detail.

Technique

Purpose

1

cDNA

To make a DNA copy of mRNA

2

Restriction
Enzymes

To cut DNA at specific points, making small
fragments

3

DNA Ligase

To join DNA fragments together

4

Vectors

To carry DNA into cells and ensure replication

5

Plasmids

Common kind of vector

6

Gene
Transfer

To
deliver a gene to a living cells

7

Genetic
Markers

To
identify cells that have been transformed

8

Replica
Plating *

To
make exact copies of bacterial colonies on an agar plate

9

PCR

To
amplify very small samples of DNA

10

DNA
probes

To
identify and label a piece of DNA containing a certain sequence

11

Shotgun
*

To
find a particular gene in a whole genome

12

Antisense
genes *

To
stop the expression of a gene in a cell

13

Gene
Synthesis

To
make a gene from scratch

14

Electrophoresis

To
separate fragments of DNA

* Additional information that is not directly
included in AS Biology. However it can help to consolidate other techniques.

Complementary
DNA (cDNA) is DNA made from mRNA. This makes use of the enzyme reverse
transcriptase, which does the reverse of transcription: it synthesises DNA
from an RNA template. It is produced naturally by a group of viruses called the retroviruses
(which include HIV), and it helps them to invade cells. In genetic engineering
reverse transcriptase is used to make an artificial gene of cDNA as shown
in this diagram.

Complementary
DNA has helped to solve different problems in genetic engineering:

It
makes genes much easier to find. There are some 70 000 genes in the human
genome, and finding one gene out of this many is a very difficult (though not
impossible) task. However a given cell only expresses a few genes, so only makes
a few different kinds of mRNA molecule. For example the b cells of the pancreas
make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can
be isolated from these cells and used to make cDNA of the insulin gene.

These
are enzymes that cut DNA at specific sites. They are properly called restriction
endonucleases because they cut the bonds in the middle of the polynucleotide
chain. Some restriction enzymes cut straight across both chains, forming blunt
ends, but most enzymes make a staggered cut in the two strands, forming sticky
ends.

The
cut ends are “sticky” because they have short stretches of
single-stranded DNA with complementary sequences. These sticky ends
will stick (or anneal) to another piece of DNA by complementary base
pairing, but only if they have both been cut with the same restriction
enzyme. Restriction enzymes are highly specific, and will only cut DNA at
specific base sequences,4-8 base
pairs long, called recognition sequences.

Restriction
enzymes are produced naturally by bacteria as a defence against viruses (they
“restrict” viral growth), but they are enormously useful in genetic
engineering for cutting DNA at precise places ("molecular scissors").
Short lengths of DNA cut out by restriction enzymes are called restriction
fragments. There are thousands of different restriction enzymes known, with
over a hundred different recognition sequences. Restriction enzymes are named
after the bacteria species they came from, so EcoR1 is from E. coli
strain R, and HindIII is from Haemophilis
influenzae.

This
enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is
commonly used in genetic engineering to do the reverse of a restriction enzyme,
i.e. to join together complementary restriction fragments.

The
sticky ends allow two complementary restriction fragments to anneal, but
only by weak hydrogen bonds, which can quite easily be broken, say by gentle
heating. The backbone is still incomplete.

DNA
ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes
and DNA ligase can therefore be used together to join lengths of DNA from
different sources.

In
biology a vector is something that carries things between species. For example
the mosquito is a disease vector because it carries the malaria parasite into
humans. In genetic engineering a vector is a length of DNA that carries
the gene we want into a host cell. A vector is needed because a length of DNA
containing a gene on its own won’t actually do anything inside a host cell.
Since it is not part of the cell’s normal genome it won’t be replicated when
the cell divides, it won’t be expressed, and in fact it will probably be
broken down pretty quickly. A vector gets round these problems by having these
properties:

It
is big enough to hold the gene we want (plus a few others), but not too big.

It
is circular (or more accurately a closed loop), so that it is less likely to
be broken down (particularly in prokaryotic cells where DNA is always
circular).

It
contains control sequences, such as a replication origin and a
transcription promoter, so that the gene will be replicated, expressed, or
incorporated into the cell’s normal genome.

It
contain marker genes, so that cells containing the vector can be
identified.

Many
different vectors have been made for different purposes in genetic engineering
by modifying naturally-occurring DNA molecules, and these are now available off
the shelf. For example a cloning vector contains sequences that cause the
gene to be copied (perhaps many times) inside a cell, but not expressed. An expression
vector contains sequences causing the gene to be expressed inside a cell,
preferably in response to an external stimulus, such as a particular chemical in
the medium. Different kinds of vector are also available for different lengths
of DNA insert:

Type
of vector

Max
length of DNA insert

Plasmid

Plasmids
are by far the most common kind of vector, so we shall look at how they are used
in some detail. Plasmids are short circular bits of DNA found naturally in
bacterial cells. A typical plasmid contains 3-5 genes and there are usually
around 10 copies of a plasmid in a bacterial cell. Plasmids are copied
separately from the main bacterial DNA when the cell divides, so the plasmid
genes are passed on to all daughter cells. They are also used naturally for
exchange of genes between bacterial cells (the nearest they get to sex), so
bacterial cells will readily take up a plasmid. Because they are so small, they
are easy to handle in a test tube, and foreign genes can quite easily be
incorporated into them using restriction enzymes and DNA ligase.

One of the most common plasmids used is the R-plasmid (or pBR322).
This plasmid contains a replication origin, several recognition sequences for
different restriction enzymes (with names like PstI
and EcoRI), and two marker genes, which confer resistance to different
antibiotics (ampicillin and tetracycline).

The
diagram below shows how DNA fragments can be incorporated into a plasmid using
restriction and ligase enzymes. The restriction enzyme used here (PstI)
cuts the plasmid in the middle of one of the marker
genes (we’ll see why this is useful later). The foreign DNA anneals with the
plasmid and is joined covalently by DNA ligase to form a hybrid vector
(in other words a mixture or hybrid of bacterial and foreign DNA).Several other products are also formed: some plasmids will
simply re-anneal with themselves to re-form the original plasmid, and some DNA
fragments will join together to form chains or circles. Theses different
products cannot easily be separated, but it doesn’t matter, as the marker
genes can be used later to identify the correct hybrid vector.

Vectors
containing the genes we want must be incorporated into living cells so that they
can be replicated or expressed. The cells receiving the vector are called host
cells, and once they have successfully incorporated the vector they are said
to be transformed. Vectors are large molecules which do not readily cross
cell membranes, so the membranes must be made permeable in some way. There are
different ways of doing this depending on the type of host cell.

Heat
Shock. Cells are incubated with the vector in a solution containing
calcium ions at 0°C. The temperature is then suddenly raised to about 40°C.
This heat shock causes some of the cells to take up the vector, though no
one knows why. This works well for bacterial and animal cells.

Electroporation.
Cells are subjected to a high-voltage pulse, which temporarily disrupts the
membrane and allows the vector to enter the cell. This is the most efficient
method of delivering genes to bacterial cells.

Viruses.
The vector is first incorporated into a virus, which is then used to infect
cells, carrying the foreign gene along with its own genetic material. Since
viruses rely on getting their DNA into host cells for their survival they
have evolved many successful methods, and so are an obvious choice for gene
delivery. The virus must first be genetically engineered to make it safe, so that it can’t
reproduce itself or make toxins. Three viruses are commonly used:

1.Bacteriophages (or phages) are viruses that infect bacteria. They
are a very effective way of delivering large genes into bacteria cells in
culture.

2.Adenoviruses are
human viruses that causes respiratory diseases including the common cold. Their
genetic material is double-stranded DNA, and they are ideal for
delivering genes to living patients in gene therapy. Their DNA is not
incorporated into the host’s chromosomes, so it is not replicated, but their
genes are expressed.

The
adenovirus is genetically altered so that its coat proteins are not synthesised,
so new virus particles cannot be assembled and the host cell is not killed.

3.Retroviruses are a
group of human viruses that include HIV. They are enclosed in a lipid membrane
and their genetic material is double-stranded RNA. On infection this RNA is
copied to DNA and the DNA is incorporated into the host’s chromosome.
This means that the foreign genes are replicated into every daughter cell.

After
a certain time, the dormant DNA is switched on, and the genes are expressed in
all the host cells.

Plant
Tumours. This method has been used successfully to transform plant
cells, which are perhaps the hardest to do. The gene is first inserted into
the Ti plasmid of the soil bacterium Agrobacterium
tumefaciens, and then plants are infected with the bacterium. The
bacterium inserts the Ti plasmid into the plant cells' chromosomal DNA and
causes a "crown gall" tumour. These tumour cells can be cultured
in the laboratory and whole new plants grown from them by micropropagation.
Every cell of these plants contains the foreign gene.

Gene
Gun. This extraordinary technique fires microscopic gold particles
coated with the foreign DNA at the cells using a compressed air gun. It is
designed to overcome the problem of the strong cell wall in plant tissue,
since the particles can penetrate the cell wall and the cell and nuclear
membranes, and deliver the DNA to the nucleus, where it is sometimes
expressed.

Micro-Injection.
A cell is held on a pipette under a microscope and the foreign DNA is
injected directly into the nucleus using an incredibly fine micro-pipette.
This method is used where there are only a very few cells available, such as
fertilised animal egg cells. In the rare successful cases the fertilised egg
is implanted into the uterus of a surrogate mother and it will develop into
a normal animal, with the DNA incorporated into the chromosomes of every
cell.

Liposomes.
Vectors can be encased in liposomes, which are small membrane
vesicles (see module 1). The liposomes fuse with the cell membrane (and
sometimes the nuclear membrane too), delivering the DNA into the cell. This
works for many types of cell, but is particularly useful for delivering
genes to cell in vivo (such as in
gene therapy).

These
are needed to identify cells that have successfully taken up a vector and so
become transformed. With most of the techniques above less than 1% of the cells
actually take up the vector, so a marker is needed to distinguish these cells
from all the others. We’ll look at how to do this with bacterial host cells,
as that’s the most common technique.

A
common marker, used in the R-plasmid, is a gene for resistance to an antibiotic
such as tetracycline. Bacterial cells taking up this plasmid can make this gene
product and so are resistant to this antibiotic. So if the cells are grown on a
medium containing tetracycline all the normal untransformed cells, together with
cells that have taken up DNA that’s not in a plasmid (99%) will die. Only the
1% transformed cells will survive, and these can then be grown and cloned on
another plate.

Replica
plating is a simple technique for making an exact copy of an agar plate. A pad
of sterile cloth the same size as the plate is pressed on the surface of an agar
plate with bacteria growing on it. Some cells from each colony will stick to the
cloth. If the cloth is then pressed onto a new agar plate, some cells will be
deposited and colonies will grow in exactly the same positions on the new plate.
This technique has a number of uses, but the most common use in genetic
engineering is to help solve another problem in identifying transformed cells.

This
problem is to distinguish those cells that have taken up a hybrid plasmid
vector (with a foreign gene in it) from those cells that have taken up the normal
plasmid. This is where the second marker gene (for resistance to ampicillin) is
used. If the foreign gene is inserted into the middle of this marker gene, the
marker gene is disrupted and won't make its proper gene product. So cells with
the hybrid plasmid will be killed by ampicillin, while cells with the normal
plasmid will be immune to ampicillin. Since this method of identification
involves killing the cells we want, we must first make a master agar plate and
then make a replica plate of this to test for ampicillin resistance.

Once
the colonies of cells containing the correct hybrid plasmid vector have been
identified, the appropriate colonies on the master plate can be selected and
grown on another plate.

The
R-plasmid with its antibiotic-resistance genes dates from the early days of
genetic engineering in the 1970s. In recent years better plasmids with different
marker genes have been developed that do not kill the desired cells, and so do
not need a replica plate. These new marker genes make an enzyme (actually
lactase) that converts a colourless substrate in the agar medium into a blue-coloured
product that can easily be seen. So cells with a normal plasmid turn blue on the
correct medium, while those with the hybrid plasmid can't make the enzyme and
stay white. These white colonies can easily be identified and transferred to
another plate. Another marker gene, transferred from jellyfish, makes a green
fluorescent protein (GFP).

Genes
can be cloned by cloning the bacterial cells that contain them, but this
requires quite a lot of DNA in the first place. PCR can clone (or amplify)
DNA samples as small as a single molecule. It is a newer technique, having been
developed in 1983 by Kary Mullis, for which discovery he won the Nobel prize in
1993. The polymerase chain reaction is simply DNA replication in a test tube. If
a length of DNA is mixed with the four nucleotides (A, T, C and G) and the
enzyme DNA polymerase in a test tube, then the DNA will be replicated many
times. The details are shown in this diagram:

Start
with a sample of the DNA to be amplified, and add the four nucleotides and
the enzyme DNA polymerase.

Normally
(in vivo) the DNA double helix
would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95°C for two
minutes. This breaks the hydrogen bonds.

DNA
polymerisation always requires short lengths of DNA (about 20 bp long)
called primers, to get it started. In
vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesised separately and added at this
stage. This means that a short length of the sequence of the DNA must
already be known, but it does have the advantage that only the part between
the primer sequences is replicated. The DNA must be cooled to 40°C to allow
the primers to anneal to their complementary sequences on the separated DNA
strands.

The
DNA polymerase enzyme can now extend the primers and complete the
replication of the rest of the DNA. The enzyme used in PCR is derived from
the thermophilic bacterium Thermus
aquaticus, which grows naturally in hot springs at a temperature of 90°C,
so it is not denatured by the high temperatures in step 2. Its optimum
temperature is about 72°C, so the mixture is heated to this temperature for
a few minutes to allow replication to take place as quickly as possible.

Each
original DNA molecule has now been replicated to form two molecules. The
cycle is repeated from step 2 and each time the number of DNA molecules
doubles. This is why it is called a chain reaction, since the number of
molecules increases exponentially, like an explosive chain reaction.
Typically PCR is run for 20-30 cycles.

PCR
can be completely automated, so in a few hours a tiny sample of DNA can be
amplified millions of times with little effort. The product can be used for
further studies, such as cloning, electrophoresis, or gene probes. Because PCR
can use such small samples it can be used in forensic medicine (with DNA taken
from samples of blood, hair or semen), and can even be used to copy DNA from
mummified human bodies, extinct woolly mammoths, or from an insect that's been
encased in amber since the Jurassic period. One problem of PCR is having a pure
enough sample of DNA to start with. Any contaminant DNA will also be amplified,
and this can cause problems, for example in court cases.

These
are used to identify and label DNA fragments that contain a specific sequence. A
probe is simply a short length of DNA (20-100 nucleotides long) with a label
attached. There are two common types of label used:

a
radioactively-labelled probe (synthesised using the isotope 32P)
can be visualised using photographic film (an autoradiograph).

a
fluorescently-labelled probe will emit visible light when illuminated with
invisible ultraviolet light. Probes can be made to fluoresce with different
colours.

Probes
are always single-stranded, and can be made of DNA or RNA. If a probe is added
to a mixture of different pieces of DNA (e.g. restriction fragments) it will
anneal (base pair) with any lengths of DNA containing the complementary
sequence. These fragments will now be labelled and will stand out from the rest
of the DNA. DNA probes have many uses in genetic engineering:

To
identify restriction fragments containing a particular gene out of the
thousands of restriction fragments formed from a genomic library. This use
is described in shotguning below.

To
identify the short DNA sequences used in DNA fingerprinting.

To
identify genes from one species that are similar to those of another
species. Most genes are remarkably similar in sequence from one species to
another, so for example a gene probe for a mouse gene will probably anneal
with the same gene from a human. This has aided the identification of human
genes.

To
identify genetic defects. DNA probes have been prepared that match the
sequences of many human genetic disease genes such as muscular dystrophy,
and cystic fibrosis. Hundreds of these probes can be stuck to a glass slide
in a grid pattern, forming a DNA microarray (or DNA chip). A
sample of human DNA is added to the array and any sequences that match any
of the various probes will stick to the array and be labelled. This allows
rapid testing for a large number of genetic defects at a time.

This is used to find one
particular gene in a whole genome, a bit like finding the proverbial needle in a
haystack. It is called the shotgun technique because it starts by
indiscriminately breaking up the genome (like firing a shotgun at a soft target)
and then sorting through the debris for the particular gene we want. For this to
work a gene probe for the gene is needed, which means at least a short part of
the gene’s sequence must be known.

These
are used to turn off the expression of a gene in a cell. The principle is very
simple: a copy of the gene to be switch off is inserted into the host genome the
“wrong” way round, so that the complementary (or antisense) strand is
transcribed. The antisense mRNA produced will anneal to the normal sense mRNA
forming double-stranded RNA. Ribosomes can’t bind to this, so the mRNA is not
translated, and the gene is effectively “switched off”.

It
is possible to chemically synthesise a gene in the lab by laboriously joining
nucleotides together in the correct order.Automated machines can now make this much easier, but only up to a limit
of about 30bp, so very few real genes could be made this way (anyway it’s
usually much easier to make cDNA). The genes for the two insulin chains (xx bp)
and for the hormone somatostatin (42 bp) have been synthesisied this way. It is
very useful for making gene probes.

This
is a form of chromatography used to separate different pieces of DNA on the
basis of their length. It might typically be used to separate restriction
fragments. The DNA samples are placed into wells at one end of a thin slab of
gel made of agarose or polyacrylamide, and covered in a buffer
solution. An electric current is passed through the gel. Each nucleotide in a
molecule of DNA contains a negatively-charged phosphate group, so DNA is
attracted to the anode (the positive electrode). The molecules have to diffuse
through the gel, and smaller lengths of DNA move faster than larger lengths,
which are retarded by the gel. So the smaller the length of the DNA molecule,
the further down the gel it will move in a given time. At the end of the run the
current is turned off.

Unfortunately
the DNA on the gel cannot be seen, so it must be visualised. There are three
common methods for doing this:

The gel can be
stained with a chemical that specifically stains DNA, such as ethidium bromide
or azure A. The DNA shows up as blue bands. This method is simple but not very
sensitive.

The DNA samples
at the beginning can be radiolabelled with a radioactive isotope such as 32P.
Photographic film is placed on top of the finished gel in the dark, and the DNA
shows up as dark bands on the film. This method is extremely sensitive.

The DNA fragments
at the beginning can be labelled with a fluorescent molecule. The DNA fragments
show up as coloured lights when the finished gel is illuminated with invisible
ultraviolet light.

This
section contains additional information that is not directly included in AS
Biology. However it can be useful to help support and consolidate GE
techniques.

We
have now looked at some of the many techniques used by genetic engineers. What
can be done with these techniques? By far the most numerous applications are
still as research tools, and the techniques above are helping geneticists to
understand complex genetic systems. Despite all the hype, genetic engineering
still has very few successful commercial applications, although these are
increasing each year. The applications so far can usefully be considered in
three groups.

Gene Products

using
genetically modified organisms (usually microbes) to produce chemicals,
usually for medical or industrial applications.

New Phenotypes

using
gene technology to alter the characteristics of organisms (usually farm
animals or crops)

The
biggest and most successful kind of genetic engineering is the production of
gene products. These products are of medical, agricultural or commercial value.
This table shows a few of the examples of genetically engineered products that
are already available.

Product

Use

Host
Organism

Insulin

human
hormone used to treat diabetes

bacteria
/yeast

HGH

human
growth hormone, used to treat dwarfism

bacteria

BST

bovine
growth hormone, used to increase milk yield of cows

bacteria

Factor
VIII

human
blood clotting factor, used to treat haemophiliacs

bacteria

Anti-thrombin

anti-blood
clotting agent used in surgery

goats

Penicillin

antibiotic,
used to kill bacteria

fungi
/ bacteria

Vaccines

hepatitis B antigen, for vaccination

yeast

AAT

enzyme used to treat cystic fibrosis and emphysema

sheep

a-glucosidase

enzyme used to treat Pompe’s
disease

rabbits

DNase

enzyme used to treat CF

bacteria

rennin

enzyme used in manufacture of cheese

bacteria /yeast

cellulase

enzyme used in paper production

bacteria

PHB

biodegradable plastic

plants

The
products are mostly proteins, which are produced directly when a gene is
expressed, but they can also be non-protein products produced by
genetically-engineered enzymes. The basic idea is to transfer a gene (often
human) to another host organism (usually a microbe) so that it will make the
gene product quickly, cheaply and ethically. It is also possible to make
“designer proteins” by altering gene sequences, but while this is a useful
research tool, there are no commercial applications yet.

Since
the end-product is just a chemical, in principle any kind of organism could be
used to produce it. By far the most common group of host organisms used to make
gene products are the bacteria, since they can be grown quickly and the product
can be purified from their cells. Unfortunately bacteria cannot not always make
human proteins, and recently animals and even plants have also been used to make
gene products. In neither case is it appropriate to extract the product from
their cells, so in animals the product must be secreted in milk or urine, while
in plants the product must be secreted from the roots. This table shows some of
the advantages and disadvantages of using different organisms for the production
of genetically-engineered gene products.

Type
of organism

Advantages

Disadvantages

Prokaryotes

(Bacteria)

No nucleus so DNA easy to modify; have plasmids;
small genome; genetics well understood; asexual so can be cloned; small
and fast growing; easy to grow commercially in fermenters; will use
cheap carbohydrate; few ethical problems.

Insulin
is a small protein hormone produced by the pancreas to regulate the blood sugar
concentration. In the disease insulin-dependent diabetes the
pancreas cells don’t produce enough insulin, causing wasting symptoms and
eventually death. The disease can be successfully treated by injection of
insulin extracted from the pancreases of slaughtered cows and pigs. However the
insulin from these species has a slightly different amino acid sequence from
human insulin and this can lead to immune rejection and side effects.

The
human insulin gene was isolated, cloned and sequenced in the 1970s, and so it
became possible to insert this gene into bacteria, who could then produce human
insulin in large amounts. Unfortunately it wasn’t that simple. In humans,
pancreatic cells first make pro-insulin, which then undergoes
post-translational modification to make the final, functional insulin. Bacterial
cells cannot do post-translational modification. Eventually
a synthetic cDNA gene was made and inserted into the bacterium E.
coli, which made pro-insulin, and the post-translational conversion to
insulin was carried out chemically. This technique was developed by Eli Lilly
and Company in 1982 and the product, “humulin” became the first
genetically-engineered product approved for medical use.

In
the 1990s the procedure was improved by using the yeast Saccharomyces
cerevisiae instead of E. coli.
Yeast, as a eukaryote, is capable of post-translational modification, so this
simplifies the production of human insulin. However another company has
developed a method of converting pig insulin into human insulin by chemically
changing a few amino acids, and this turns out to be cheaper than the genetic
engineering methods. This all goes to show that genetic engineers still have a
lot to learn.

HGH
is a protein hormone secreted by the pituitary gland, which stimulates tissue
growth. Low production of HGH in childhood results in pituitary dwarfism.
This can be treated with HGH extracted from dead humans, but as the treatment
caused some side effects, such as Creutzfeldt-Jacod disease (CJD), the treatment
was withdrawn. The HGH gene has been cloned and an artificial cDNA gene has been
inserted into E. coli. A signal
sequence has been added which not only causes the gene to be translated but also
causes the protein to be secreted from the cell, which makes purification much
easier. This genetically engineered HGH is produced by Genentech and can
successfully restore normal height to children with HGH defficiency.

This is a growth hormone produced by cattle. The gene has
been cloned in bacteria by the company Monsanto, who can produce large
quantities of BST. in the USA cattle are often injected with BST every 2 weeks,
resulting in a 10% increase in mass in beef cattle and a 25% increase in milk
production in dairy cows. BST was tested in the UK in 1985, but it was not
approved and its use is currently banned in the EU. This is partly due to public
concerns and partly because there is already overproduction of milk and beef in
the EU, so greater production is not necessary.

Rennin
is an enzyme used in the production of cheese. It is produced in the stomach of
juvenile mammals (including humans) and it helps the digestion of the milk
protein caesin by solidifying it so that is remains longer in the stomach.
Traditionally the cheese industry has used rennin obtained from the stomach of
young calves when they are slaughtered for veal, but there are moral and
practical objections to this source. Now an artificial cDNA gene for rennin has
been made from mRNA extracted from calf stomach cells, and this gene has been
inserted into a variety of microbes such as the bacterium E.
coli and the fungus Aspergillus niger.
The rennin extracted from these microbes has been very successful and 90% of all
hard cheeses in the UK are made using microbial rennin. Sometimes (though not
always) these products are labelled as “vegetarian cheese”.

AAT is a human protein made in the liver and
found in the blood. As the name suggests it is an inhibitor of protease enzymes
like trypsin and elastase. There is a rare mutation of the AAT gene (a single
base substitution) that causes AAT to be inactive, and so the protease enzymes
to be uninhibited. The most noticeable effect of this in the lungs, where elastase
digests the elastic tissue of the alveoli, leading to the lung disease emphysema.
This condition can be treated by inhaling an aerosol spray containing AAT so
that it reaches the alveoli and inhibits the elastase there.

AAT
for this treatment can be extracted from blood donations, but only in very small
amounts. The gene for AAT has been found and cloned, but AAT cannot be produced
in bacteria because AAT is glycoprotein, which means it needs to have
sugars added by post translational modification. This kind of modification can
only be carried out by animals, and AAT is now produced by genetically-modified
sheep. In order to make the AAT easy to extract, the gene was coupled to a
promoter for the milk protein b-lactoglubulin. Since this promoter is only
activated in mammary gland cells, the AAT gene will only be expressed in mammary
gland cells, and so will be secreted into the sheep's milk. This makes it very
easy to harvest and purify without harming the sheep. The first transgenic sheep
to produce AAT was called Tracy, and she was produced by PPL Pharmaceuticals in
Edinburgh in 1993. This is how Tracy was made:

A
female sheep is given a fertility drug to stimulate her egg
production, and several mature eggs are collected from her ovaries.

The
eggs are fertilised in vitro.

A
plasmid is prepared containing the gene for human AAT
and the promoter sequence for b-lactoglobulin. Hundreds of copies of
this plasmid are microinjected into the nucleus of the fertilised
zygotes. Only a few of the zygotes will be transformed, but at this
stage you can’t tell which.

The
zygotes divide in vitro
until the embryos are at the 16-cell stage.

The
16-cell embryos are implanted into the uterus of surrogate mother
ewes. Only a few implantations result in a successful pregnancy.

Test
all the offspring from the surrogate mothers for AAT production in
their milk. This is the only way to find if the zygote took up the AAT
gene so that it can be expressed. About 1 in 20 eggs are successful.

Collect
milk from the transgenic sheep for the rest of their lives. Their milk
contains about 35 g of AAT per litre of milk. Also breed from them in
order to build up a herd of transgenic sheep.

This
means altering the characteristics of organisms by genetic engineering. The
organisms are generally commercially-important crops or farm animals, and the
object is to improve their quality in some way. This can be seen as a high-tech
version of selective breeding, which has been used by humans to alter and
improve their crops and animals for at least 10 000 years. Nevertheless GMOs
have turned out to be a highly controversial development. We don’t need to
study any of these in detail, but this table gives an idea of what is being
done.

Organism

Modification

Long
life tomatoes

There
are two well-known projects, both affecting the gene for the enzymepolygalactourinase (PG), a pectinase that softens fruits as
they ripen. Tomatoes that make less PG ripen more slowly and retain more
flavour. The American “Flavr Savr” tomato used antisense technology
to silence the gene, while the British Zeneca tomato disrupted the gene.
Both were successful and were on sale for a few years, but neither is
produced any more.

Insect-resistant
crops

Genes
for various powerful protein toxins have been transferred from the
bacterium Bacillus thuringiensis to crop plants including maize, rice and
potatoes. These Bt toxins are thousands of times more powerful
than chemical insecticides, and since they are built-in to the crops,
insecticide spraying (which is non-specific and damages the environment)
is unnecessary.

Virus-resistant
crops

Gene
for virus coat protein has been cloned and inserted into tobacco, potato
and tomato plants. The coat protein seems to “immunise” the plants,
which are much more resistant to viral attack.

Herbicide
resistant crops

The gene
for resistance to the herbicide BASTA has been transferred from Streptomyces
bacteria to tomato, potato, corn, and wheat plants, making them
resistant to Basta. Fields can safely be sprayed with this herbicide,
which will kill all weeds, but the crops. However, this means using more
agrochemicals, not less.

Pest-resistant
legumes

The
gene for an enzyme that synthesises a chemical toxic to weevils has been
transferred from Bacillus
bacteria to The Rhizobium
bacteria that live in the root nodules of legume plants. These root
nodules are now resistant to attack by weevils.

Nitrogen-fixing
crops

This
is a huge project, which aims to transfer the 15-or-so genes required
for nitrogen fixation from the nitrogen-fixing bacteria Rhizobium
into cereals and other crop plants. These crops would then be able to
fix their own atmospheric nitrogen and would not need any fertiliser.
However, the process is extremely complex, and the project is nowhere
near success.

Crop
improvement

Proteins
in some crop plants, including wheat, are often deficient in essential
amino acids (which is why vegetarians have to watch their diet so
carefully), so the protein genes are being altered to improve their
composition for human consumption.

Mastitis-resistant
cattle

The
gene for the enzyme lactoferrin, which helps to resists the infection
that causes the udder disease mastitis, has been introduced to Herman
– the first transgenic bull. Herman’s offspring inherit this gene,
do not get mastitis and so produce more milk.

Tick-resistant
sheep

The
gene for the enzyme chitinase, which kills ticks by digesting their
exoskeletons, has bee transferred from plants to sheep. These sheep
should be immune to tick parasites, and may not need sheep dip.

Fast-growing
sheep

The
human growth hormone gene has been transferred to sheep, so that they
produce human growth hormone and grow more quickly. However they are
more prone to infection and the females are infertile.

Fast-growing
fish

A
number of fish species, including salmon, trout and carp, have been
given a gene from another fish (the ocean pout) which activates the
fish’s own growth hormone gene so that they grow larger and more
quickly. Salmon grow to 30 times their normal mass at 10 times the
normal rate.

Environment
cleaning microbes

Genes
for enzymes that digest many different hydrocarbons found in crude oil
have been transferred to Pseudomonas
bacteria so that they can clean up oil spills.

This
is perhaps the most significant, and most controversial kind of genetic
engineering. It is also the least well-developed. The idea of gene therapy is to
genetically alter humans in order to treat a disease. This could represent the
first opportunity to cure incurable diseases.Note that this is quite different from using
genetically-engineered microbes to produce a drug, vaccine or hormone to treat a
disease by conventional means. Gene therapy means altering the genotype of a
tissue or even a whole human.

Cystic
fibrosis (CF) is the most common genetic disease in the UK, affecting about 1 in
2500. It is caused by a mutation in the gene for protein called CFTR (Cystic
Fibrosis Transmembrane Regulator). The gene is located on chromosome 7,
and there are actually over 300 different mutations known, although the most
common mutation is a deletion of three bases, removing one amino acid out of
1480 amino acids in the protein. CFTR is a chloride ion channel protein found in
the cell membrane of epithelial (lining) tissue cells, and the mutation stops
the protein working, so chloride ions cannot cross the cell membrane.

Chloride
ions build up inside these cells, which cause sodium ions to enter to balance
the charge, and the increased concentration of the both these ions inside the
epithelial cells decreases the osmotic potential. Water is therefore retained
inside the cells, which means that the mucus secreted by these cells is drier
and more sticky than normal. This sticky mucus block the tubes into which it is
secreted, such as the small intestine, pancreatic duct, bile duct, sperm duct,
bronchioles and alveoli.

These
blockages lead to the symptoms of CF: breathlessness, lung infections such as
bronchitis and pneumonia, poor digestion and absorption, and infertility. Of
these symptoms the lung effects are the most serious causing 95% of deaths. CF
is always fatal, though life expectancy has increased from 1 year to about 20
years due to modern treatments. These treatments include physiotherapy many
times each day to dislodge mucus from the lungs, antibiotics to fight
infections, DNAse drugs to loosen the mucus, enzymes to help food digestion and
even a heart-lung transplant.

Given
these complicated (and ultimately unsuccessful) treatments, CF is a good
candidate for gene therapy, and was one of the first diseases to be tackled this
way. The gene for CFTR was identified in 1989 and a cDNA clone was made soon
after. The idea is to deliver copies of this good gene to the epithelial cells
of the lung, where they can be incorporated into the nuclear DNA and make
functional CFTR chloride channels. If about 10% of the cells could be corrected,
this would cure the disease.

Two
methods of delivery are being tried: liposomes and adenoviruses, both delivered
with an aerosol inhaler, like those used by asthmatics. Clinical trials are
currently underway, but as yet no therapy has been shown to be successful.

Severe
Combined Immunodefficiency Disease (SCID) is a rare genetic disease that affects
the immune system. It is caused by a mutation in the gene for the enzyme adenosine
deaminase (ADA). Without this enzyme white blood cells cannot be made, so
sufferers have almost no effective immune system and would quickly contract a
fatal infection unless they spend their lives in sterile isolation (SCID is also
known as “baby in a bubble syndrome”). Gene therapy has been attempted with
a few children in the USA and UK by surgically removing bone marrow cells (which
manufacture white blood cells in the body) from the patient, transfecting them
with a genetically-engineered virus containing the ADA gene, and then returning
the transformed cells to the patient. The hope is that these transformed cells
will multiply in the bone marrow and make white blood cells.
The trials are still underway, so the success is unknown.

Gene
therapy is in its infancy, and is still very much an area of research rather
than application. No one has yet been cured by gene therapy, but the potential
remains enticing. Gene therapy need not even be limited to treating genetic
diseases, but could also help in treating infections and environmental
diseases:

White
blood cells have be genetically modified to produce tumour necrosis factor (TNF),
a protein that kills cancer cells, making these cells more effecting against
tumours.

Genes
could be targeted directly at cancer cells, causing them to die, or to
revert to normal cell division.

White
blood cells could be given antisense genes for HIV proteins, so that if the
virus infected these cells it couldn’t reproduce.

It
is important to appreciate the different between somatic cell therapy
and germ-line therapy.

Somatic
cell therapy means genetically altering specific body (or somatic) cells,
such as bone marrow cells, pancreas cells, or whatever, in order to treat
the disease. This therapy may treat or cure the disease, but any genetic
changes will not be passed on their offspring.

Germ-line
therapy means genetically altering those cells (sperm cells, sperm precursor
cell, ova, ova precursor cells, zygotes or early embryos) that will pass their genes
down the “germ-line” to future generations. Alterations to any of these
cells will affect every cell in the resulting human, and in
all his or her descendants.

Germ-line
therapy would be highly effective, but is also potentially dangerous (since the
long-term effects of genetic alterations are not known), unethical (since it
could easily lead to eugenics) and immoral (since it could involve altering and
destroying human embryos). It is currently illegal in the UK and most other
countries, and current research is focusing on somatic cell therapy only. All
gene therapy trials in the UK must be approved by the Gene Therapy Advisory
Committee (GTAC), a government body that reviews the medical and ethical grounds
for a trial. Germ-line modification is allowed with animals, and indeed is the
basis for producing GMOs.